Project summary: Our research objective is to define the mechanistic basis of Hsp104, a protein disaggregase and hexameric AAA+ (ATPases Associated with diverse Activities) protein from yeast, which remains poorly understood. Hsp104 couples ATP hydrolysis to the dissolution and reactivation of diverse proteins trapped in disordered aggregates, toxic preamyloid oligomers, amyloids, and prions. Hsp104 is the only factor known to dissociate ?-synuclein (?-syn) oligomers and amyloids connected with Parkinson's disease (PD) and rescue ?-syn-induced neurodegeneration in the substantia nigra of a rat PD model. However, Hsp104 activity is limited against ?-syn and very high Hsp104 concentrations are needed for optimal effects. Thus, we engineered potentiated Hsp104 variants, which dissolve fibrils formed by neurodegenerative disease proteins such as TDP-43, FUS, and ?-syn, and mitigate neurodegeneration in the metazoan nervous system at concentrations where Hsp104 is inactive. Curiously, Hsp104 is absent from metazoa. Thus, Hsp104 and potentiated variants could represent a disruptive technology to enhance proteostasis to counter neurodegenerative disease and enable purification of irksome, aggregation-prone proteins for valuable basic or pharmaceutical purposes. However, these endeavors are frustrated by a limited mechanistic understanding of Hsp104, which despite intense investigation remains stalled at a low level of resolution. Three critical barriers impede our understanding of Hsp104. First, we do not understand how Hsp104 selects clients for disaggregation, which limits our ability to tailor Hsp104 activity for specific substrates. This issue is pernicious because potentiated Hsp104 variants can have damaging, off-target effects due to promiscuous activity, which could restrict therapeutic or biotechnological applications. Second, Hsp104 sequence space remains largely unexplored. It is unclear whether natural Hsp104 orthologues exist with divergent enhanced or selective activity against neurodegenerative disease substrates. Third, there is no atomic structure of the Hsp104 hexamer and conflicting cryo-electron microscopy reconstructions have confused the field. Based on our preliminary data, we hypothesize that: (1) potentiated Hsp104 variants can be engineered to be more substrate specific to avoid damaging off-target effects; (2) natural Hsp104 orthologues exist with enhanced activity against neurodegenerative disease substrates and minimal off-target effects; and (3) large structural changes in Hsp104 hexamers upon ATP hydrolysis drive protein disaggregation. Thus, we will meet three aims: (1) Define potentiated Hsp104 variants with enhanced substrate selectivity; (2) Define conserved and divergent activities of natural Hsp104 orthologues; (3) Define high-resolution structural changes in Hsp104 and potentiated variants that drive protein disaggregation. In this way, we will secure a high- resolution mechanistic view of Hsp104, which will empower the engineering of new Hsp104 nanomachines with selective potentiated activity for key applications in biotechnology and medicine.